Bartonella bacilliformis (Bb) is a highly virulent, sand fly-borne, gram-negative bacterium that causes bartonellosis (Carrin's disease) in humans. Bartonellosis is reemerging in endemic regions (high Andes) and expanding into non-endemic areas (i.e., lower altitudes and more diverse habitats) of Ecuador, Colombia and Peru. The at-risk population is estimated at 1.7 million people in a 56,000 square-mile area of S. America. Incidence rates of 12.7 / 100 person years have been reported in endemic regions. Bb infections can be life threatening, with fatality rates of 40-88%, if left untreated, and 10% fatalty following treatment with antimicrobials. Pediatric populations are especially at risk. In non-endemic regions, the disease manifests as an acute illness with an ~80% reduction in erythrocyte hematocrit (Oroya fever), whereas in endemic regions, angiomatous skin lesions (verruga peruana) and chronic bacteremia prevail, creating a human reservoir of Bb. Little is known about Bb's molecular pathogenesis, virulence determinants and its association with the sand fly vector, Lutzomyia verrucarum. To address this dearth of information, we propose to examine Bb's relationship with L. verrucarum, by live imaging techniques. We also propose to examine the role of three putative virulence determinants [i.e., flagellin (FlaA), heme-binding protein C (HbpC) and the invasion-associated locus B (IalB) protein] in Bb's colonization of L. verrucarum. We hypothesize that Bb initially infects the insect's midgut epithelium upon ingestion of a contaminated blood-meal. From this focus of infection, the hemocoel and eventually the salivary glands become infected, owing to the highly invasive nature of Bb, and that the resulting infection is life-long and not trans-ovarially transmitted. Finally, we hypothesze that Bb mutants which lack the aforementioned virulence factors will be impaired in their ability to colonize, replicate and/or spread in the sand fly. To address these hypotheses, we propose the flowing aims: In Aim 1, we will analyze colonization and migration of a low-passage, GFP+ Bb strain in sand flies. The first goal is to generate a low-passage Bb strain that contains a stable, GFP-expressing insert. The second goal is to follow the anatomical locations of the GFP+ Bb strain in the sand fly over time, by confocal microscopy. In Aim 2, we will generate hbpC, ialB and flaA mutants in low-passage Bb strains. The first goal is to generate low-passage, Bb strains whose hbpC, ialB and flaA virulence determinants have been mutagenized by a novel technique. The second goal is to create corresponding, complemented strains by allelic restoration. In Aim 3, we will compare wild-type (WT), mutant and complemented Bb strains for the ability to colonize and persist in sand flies. The first goal is to compare the reltive ability of these Bb strains to colonize sand flies. The second goal is to compare the ability of these strains to replicate and persist in the sand fly. Aim 3 results will be used to address molecular Koch's postulates for these genes, as it relates to the sand fly vector. The results of the study are expected to provide a solid foundation for eventually developing control strategies designed to interrupt the sand fly-Bb association.